Laboratory Techniques in Immunology Practical Applications of Immunology

ANTIGEN-ANTIBODY INTERACTIONS Many diagnoses in infectious disease and pathology would not be possible without laboratory procedures that identify antibodies or antigens in the patient Interaction of antigen and antibody occurs in vivo, and in clinical settings it provides the basis for all serologically based tests. The formation of immune complexes produces a visible reaction that is the basis of precipitation and agglutination tests.

Diagnostic Immunology Many tests based have been developed to determine the presence of antibodies or antigens in a patient to diagnose infectious diseases and pathology Clinical sensitivity - ability of a test to provide a positive result if the patient has the disease (no false negatives). Clinical specificity – ability of a test to give a negative result if the patient does not have disease (no false positives).

Lattice formation not required Labeled immunoassay Lattice formation not required

FLUORESCENT ANTIBODY TESTS The direct fluorescent antibody test (DFA) is used to detect and localize antigen in the patient. The tissue sample to be tested is treated with antibodies against that particular antigen that have been labeled with a fluorescent dye. If the antigen is present in the tissues, the fluorescent-labeled antibodies will bind, and their binding can be detected with a fluorescence microscope. Variations of this test are used to diagnose respiratory syncytial virus, herpes simplex- 1 and -2, and Pneumocystis infections.

Excitation/Emission Spectra

Immunofluorescence Qualitative Direct Ab to tissue Ag is labeled with a fluorochrome. Ag Y Fluorochrome Labeled Ab Tissue Section Direct Fluorescent-Antibody Tests Direct fluorescent-antibody tests are used to identify specific microorganisms (antigens). Antibodies directed against antigens on the surface of a specific microorganism are labeled with fluorescent dye. Fluorescent antibodies are incubated with the sample and antigen-specific binding allowed to occur Excess and non-specifically attached antibodies are washed from the sample The sample is viewed with a fluorescence viewer, whether a fluorescence microscope or plate reader or even fluorescence-activated flow cytometer

Direct Immunofluorescence

The indirect fluorescent antibody test (IFA) is used to detect pathogen-specific antibodies in the patient. In this case, a laboratory-generated sample of infected tissue is mixed with serum from the patient. A fluorescent dye-labeled anti-immunoglobulin (raised in animals) is then added. If binding of antibodies from the patient to the tissue sample occurs, then the fluorescent antibodies can be bound, and fluorescence can be detected in the tissue by microscopy. This technique is used to detect antinuclear antibodies, anti-dsDNA antibodies, antithyroid antibodies, antiglomerular basement-membrane antibodies, and anti-Epstein-Barr virus viral-capsid antigen antibodies.

Immunofluorescence Indirect Ab to tissue Ag is unlabeled. Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab Indirect Fluorescent-Antibody Tests Indirect fluorescent-antibody tests are used to demonstrate the presence of antibodies against a specific antigen in serum. Antigen or the microorganism itself is incubated with the patient's serum

Indirect Immunofluorescence

In suspected Graves' disease the first-line test is antibodies against TSH receptors (TRAb), which occur with a prevalence of 90 to 100 % (ELISA). The detection of antibodies against TPO can support the diagnosis (ELISA or IIFT). In Hashimoto's thyroiditis serological antibodies against TPO are detectable with a prevalence of up to 90 %, while antibodies against TG are occur in 60 to 70 % of cases.

RADIOIMMUNOASSAY (RIA) AND ENZVME-LINKED IMMUNOABSORBENT ASSAY (EIA OR ELISA) RIA and ELISA are extremely sensitive tests (as little as 10-9 g of material can be detected) that are common in medical laboratories. They can be used to detect the presence of hormones, drugs, antibiotics, serum proteins, infectious disease antigens, and tumor markers. Both tests are conducted similarly, but the RIA uses the detection of a radiolabeled product and the ELISA detects the presence of enzyme-mediated color changes in a chromogenic substrate.

ELISA In the screening test for HIV infection, the ELISA is used, with the p24 capsid antigen from the virus coated on to microtiter plates. The serum from the patient is then added, followed by addition of an enzyme-labeled antihuman immunoglobulin. Finally, the enzyme substrate is added, and the production of a color change in the well can be observed if all reagents bind one another in sequence.

Double Antibody ELISA(direct) Ag detection Immobilize Ab Incubate with sample Add labeled antibody Amount of labeled Ab bound is proportional to the amount of Ag in the sample Solid Phase Y Ag Immobilized Ag in Patient’s sample Labeled Ab Quantitative

Indirect ELISA Ab detection Immobilize Ag Incubate with sample Add labeled anti-Ig Amount of labeled Ab bound is proportional to amount of Ab in the sample Solid Phase Y Ag Immobilized Ab in Patient’s sample Labeled Anti-Ig Quantitative

Applications Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test or West Nile Virus) and also for detecting the presence of antigen. It has also found applications in the food industry in detecting potential food allergens such as milk,peanuts,walnuts,almonds, and eggs. 19

FLUORESCENCE-ACTIVATED CELL SORTER This procedure is used to rapidly analyze cell types in a complex mixture and sort them into different populations based on their binding to specific fluorescent dyes. By using antibodies against specific cell-surface markers conjugated to different fluorescent dyes, it is possible to analyze the relative numbers of cells present in a specific tissue location.

As cells pass through the apparatus in a single file, a computer-generated graph is produced, plotting the intensity and color of fluorescence of each cell along the axes. Each dot on the graph reflects the passage of a cell with a certain level and color of fluorescence, so the darkly dotted areas of the graph reflect the presence of many cells of similar attributes. Cells with high fluorescence from both dyes will therefore be found in the top right quadrant.

Flow Cytometry Cells in suspension are labeled with fluorescent tag. Direct or indirect fluorescence Cells analyzed on a flow cytometer. www.abcam.com

Principle of Flow Cytometry Cell sample labeled with appropriate fluorescent Abs Cells in suspension are passed through machine in single file in a stream of fluid Stream is focused through one or more laser beams, measuring light scatter and fluorescence characteristics Fluorescence detected by photomultiplier tubes (PMTs) Signals sent to computer for analysis

Flow Cytometry Interpretation One Parameter Histogram Red Fluorescence Intensity Green Fluorescence Intensity Two Parameter Histogram Unstained cells FITC-labeled cells Number of Cells Green Fluorescence Intensity

Myeloma

Common Applications of Flow Cytometry in Immunology Phenotype of cell, surface molecules Intracellular cytokine staining Antigen specificity Cell proliferation (e.g. CFSE, BrdU incorporation) Cell sorting Apoptosis analysis Cytotoxicity assays Phagocytosis assays Cell cycle analysis (DNA content analysis) Cell signalling molecules, Calcium flux assays Organelle-specific studies (e.g. lysosome) Cellular transport assays Transfection efficiencies